INTRODUCTION- The accurate replication of duplex DNA requires the coordinated activity of many types of proteins. We are using a multienzyme system of phage T4 proteins to determine the nature of the proteins, enzymatic reactions, and protein-protein interactions that catalyze and control this intricate and essential process. T4 DNA polymerase, attached to a sliding clamp protein, catalyzes DNA synthesis on both the leading and lagging strands. The gene 41 helicase moves 5' to 3' on the lagging strand template, opening the duplex ahead of the leading strand polymerase, and interacting with the primase to allow it to make the RNA primers initiating lagging strand synthesis. Although the helicase can load on nicked and forked DNA by itself, its loading is greatly accelerated by the 59 helicase loading protein. The RNA primers and adjacent DNA are ultimately removed by a T4 encoded 5' to 3' nuclease (T4 RNase H), and following gap repair, the adjacent fragments are joined by T4 DNA ligase. T4 ssDNA binding protein (32 protein) coats the ssDNA at the fork, binds directly to the polymerase, primase, helicase loading protein, and RNaseH, and plays an essential role in coordinating the reactions of these proteins. HELICASE, HELICASE LOADER, AND 32 PROTEIN- We have continued our studies of the mechanism by which the 59 helicase-loading protein binds to 32 protein, and loads the helicase at a replication fork. We have previously shown that 59 protein binds preferentially to forked DNA. In contrast, linear ssDNA is the preferred substrate for 32 protein. We find that 32 protein has a much higher affinity for ssDNA regions on the lagging strand of a fork than for the ssDNA regions on the leading strand. Moreover, on linear DNA 32 protein binds more tightly to ssDNA adjacent to the 3' end of an annealed complementary strand than to ssDNA near the 5' end. These findings are compatible with a model in which the array of contiguous 32 protein is assembled from the 5' to the 3' end of a single-strand, consistent with the role of 32 protein in covering the ssDNA that is unwound by the helicase moving 5' to 3' on the lagging strand of the fork. 59 protein promotes the binding of 32 protein on forks too short for cooperative 32 binding, and may play a role in loading the first 32 protein on the ssDNA behind each new lagging strand primer. The two C-terminal residues of 59 protein are necessary for this reaction, but are not needed to bind 32 protein in the absence of DNA. It is possible that these residues, lysine-216 and tyrosine-217, help to hold the fork arm in a configuration that is more accessible to 32 protein. In collaboration with Eric Miller (North Carolina State University) we have cloned the genes for the 41 helicase and 59 helicase loading protein from the KVP40 vibriophage that is related to T4. We will use the purified KVP40 proteins, and chimeric KVP40-T4 proteins to help define the regions of the helicase and helicase loading protein that interact with each other. In collaboration with Timothy Mueser (University of Toledo), we are continuing our efforts to get crystal structures of 59 protein and T4 RNaseH bound to DNA. T4 phage replication begins with synthesis from one of several origins, but later T4 replication is initiated at forks in recombination molecules. Previous studies with mutants in gene 59 established that the helicase loader was essential for recombination-dependent replication, but suggested it was not needed for the early origin-dependent synthesis. However, we found that 59 protein strongly stimulated replication in vitro from one of the T4 origins. We have now constructed a T4 phage with a complete deletion of gene 59, and shown that it is severely defective in DNA synthesis and produces a very low phage yield. We are currently characterizing the nature of the replication in the absence of 59 protein. Replication of the T4 59 deletion phage can be restored by providing 59 protein from a plasmid. We have begun a genetic screen to identify essential regions of 59 protein by selecting for plasmid mutants that fail to restore replication of the T4 gene 59 deletion. STRUCTURE OF REPLICATION FORKS- We are collaborating with Jack Griffith (University of North Carolina) to characterize the path of DNA and the structure of proteins on T4 replication forks by electron microscopy. On the lagging strand of the fork there will be ssDNA behind the nascent fragment, and a second region of ssDNA ahead of those fragments that are still being elongated by polymerase. Bruce Albert's proposed that the new lagging strand fragment folds into a loop to allow the lagging strand polymerase to be in contact with the leading strand proteins. In confirmation of this "trombone model" we found that there was a fully duplex loop associated with the replisome in 56% of the rolling circle molecules. However, the ssDNA segments at the fork, bound by T4 32 protein and 59 helicase loading protein, appear to be organized into highly compact structures ("bobbins") within the replisome complex, in contrast to the extended ssDNA envisioned in the original model. These bobbins are not present in reactions where lagging strand synthesis is prevented by omitting primase, are less compact in reactions without the 59 helicase loader, and can be disrupted by elevating the level of salt to non-physiological conditions. We have used streptavidin beads attached to a short rigid dsDNA as visible pointers to show biotin-tagged polymerase, helicase, 59 helicase loading protein, and 32 protein within the complexes on molecules with extensive replication. We are now analyzing large numbers of both native and partially disrupted complexes to investigate where within the complexes the biotin-tagged proteins are located, and how the ssDNA and proteins are organized within the "bobbins".